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Old 11-25-2016, 06:23 AM   #1
HelenaSC
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Location: Spain

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Default QIAseq targeted DNA Panel Question

Hi alla!
Anyone has been working with QIAseq targeted DNA Panel??

It is our first trial and I'm not very glad with the libraries profiles on Bioanalyzer... We have performed a first experiment with two fresh frozen samples (tubes 1 and 2, input 50 ng DNA) and their FFPE partners (at 2 different Input amount; 100 ng and 250 ng, tubes 3, 4, 5 i 6).

What do you think of the library profile? It seems that there are some large fragments in our libraries...any idea?For the fresh frozen it could be an issue related to the PCR...but in the FFPE?? (It doesn't seem to be beads, in my opinion).

Here are the profiles. Thanks a lot in advance!
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Old 01-17-2017, 09:17 AM   #2
jvdmeule
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Hi HelenaSC,

We have just performed a first trial using the QIAseq targeted DNA panel (actionable tumor panel). I also see that we have large fragments in our libraries.
Have you find why these large fragments were present? And have you found a solution?

Many thanks!
Joni
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Old 01-18-2017, 06:37 AM   #3
HelenaSC
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Hi Joni,

Yes, the larger fragments are supposed to be overamplification. I made a mistake using the recommendation from the tables, i choosed the wrong conditions regarding the number of cycles in relation to tje number of primers from our panel... Anyway, I repeated the protocol with the correct conditions and, altough some of the samples resulted in much better libraries profile, others just remained the same...

We tried to Run these samples together on a Run and we are experiencing some problems, it seems that the MiSeq was able to detect the clusters (1400) but it wasn't able to give us any more results, and none of the clusters has good passing filter QC...strange... Here is an image from the BySpace, maybe it is more easy to understand...Did you performed a Run?

Imagen1.jpg

Imagen2.jpg
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